PROJECT SUMMARY Rejection remains the major cause of allograft loss and its frequency and severity are exacerbated by peri- operative ischemia/reperfusion injury (IRI) of the graft. A mechanistic explanation of this connection is that IRI of graft endothelial cells (EC) enhances their ability to stimulate the host immune system, exacerbating rejection. Ex vivo normothermic perfusion (EVNP) is an experimental approach, pioneered for renal transplantation by our collaborators at the University of Cambridge, to reduce graft injury by improving the metabolic state of the tissue. We hypothesize that this procedure can simultaneously be used to ameliorate IRI and reduce rejection by delivery of siRNA to graft EC prior to organ implantation. We have selected tumor necrosis factor receptor 1 (TNFR1) and its key adaptor protein, TRADD, as molecular targets for siRNA knockdown in these experiments because these molecules mediate tumor necrosis factor-? responses of EC that promote inflammation and cell death, important processes in renal IRI, but the goal of this R21 is limited to optimization of the effect of siRNA knockdown. siRNA delivery in the clinic is currently limited by incomplete extent and short duration of knockdown, by inability to efficiently target the cells of interest and by effects of inappropriate cellular targets. Here, we propose proof-of-principle studies to demonstrate that antibody- conjugated, degradable polymer nanoparticles (NP) can be designed to overcome these limitations. Our specific aims are: (1) to optimize formulation of multifunctional, degradable NPs using both established and novel degradable polymers for siRNA loading, siRNA sustained release and mAb conjugation; (2) to identify the best EC specific mAb or combination of mAb for binding and uptake of conjugated NP by cultured human EC under flow in vitro at Yale and in deceased donor kidneys undergoing EVNP, the latter to be performed by our collaborators at the University of Cambridge with resources independent of this R21 application; Successful completion of these aims will provide proof-of-concept for an innovative approach to reduce allograft rejection by ex vivo targeting the graft with novel drug delivery vehicles, sparing the host from the need to excessive immunosuppression.